The initial synthesis of hormones and neuropeptides as large polypeptide precursors following synthesis is a widespread phenomenon that has been demonstrated in organisms from yeast to mammals. The post-translational liberation of bioactive molecules from their propeptide structures requires a host of processing enzymes that also appear to be conserved across eucaryotes.
The first step in the production of mature peptides and hormones, endoproteolytic cleavage, appears to require sequence specificity. Endoproteases specific for several recurrent amino acid sequence motifs identified in propeptide and prohormone polypeptides have been isolated and their substrate recognition sites explored. [Checler, et al., "Purification and characterization of a novel neurotensin-degrading peptidase from rat brain synaptic membranes," J. Biol. Chem. 261, 11274-11281 (1986); Clamagirand, et al., "Partial purification and functional properties of an endoprotease from bovine neurosecretory granules cleaving proocytocin/neurophysin peptides at the basic amino acid doublet," Biochem. 26, 6018-6023 (1987); Gluschankof, et al., "Role of peptide substrate structure in the selective processing of peptide prohormones at basic amino acid pairs by endoproteases," FEBS Lett. 234, 149-152 (1988)] Examples include endopeptidases specific for paired basic residues (commonly Lys-Arg or Arg-Arg), and cleavage sites characterized by a single proline. Monobasic residue recognition sites have also been found to attract specific proteolytic enzymes which hydrolyze peptide bonds either on the amino or carboxyl terminal side of a single arginine or lysine.
Physiological propeptides and prohormones often contain multiple cleavage sites, as well as amino acid sequences that comprise the recognition site yet do not undergo hydrolysis. One aspect of the putative sequence-specific endopeptidases is their ability to hydrolyze selected peptide bonds while leaving other consensus sites uncleaved. Furthermore, tissue-specific processing events demonstrate the ability of endoproteases to attack particular peptide bonds in one tissue but not others. [Loh, et al., "Proteolysis in neuropeptide processing and other neural functions," Annu. Rev. Neuro. 7, 189-222 (1984)] Clearly, additional parameters determine substrate recognition by endopeptidases, and most investigators concede that higher order structure must play a critical role.
In many experimental systems designed to study proteolytic processing, primary sequence simply cannot account for the selectivity of peptide bonds hydrolyzed by a given enzyme. For this reason, a number of reports documenting endoprotease substrate specificity acknowledge an apparent role played by secondary structure. [Beinfeld, et al., "Characterization of an endoprotease from rat small intestinal mucosal secretory granules which generates somatostatin-28 from prosomatostatin by cleavage after a single arginine residue," J. Biol. Chem. 264, 4460-4465 (1989), Gluschankof et al., supra (1988)] More recently, the structural features which govern peptide processing have been examined by assaying shortened synthetic analogues or precursor polypeptides modified by site-directed mutagenesis of cDNAs. [Brakch, et al., "Processing endoprotease recognizes a structural feature at the cleavage site of peptide prohormones," J. Biol. Chem. 264, 15912-15916 (1989); Docherty, et al., "Proinsulin endopeptidase substrate specificities defined by site-directed mutagenesis of proinsulin," J. Biol. Chem. 264, 18335-18339 (1989); Gomez, et al., "Site-specific mutagenesis identifies amino acid residues critical in prohormone processing," EMBO J. 8, 2911-2916 (1989); and Thorne, et al., "An in vivo characterization of the cleavage site specificity of the insulin cell prohormone processing enzymes," J. Biol. Chem. 265, 8436-8443 (1990)] Studies of this nature usually succeed only in identifying a specific residue at or nearby a cleavage site that influences substrate susceptibility, and the contribution made by the amino acid to the overall structure are of the full length substrate are not assessed.
A statistical analysis that revealed the high probability of .beta.-turns at cleavage sites characterized by dibasic residues served to direct new attention to this particular structural motif. [Rholam, et al., "Precursors for peptide hormones share common secondary structures forming features at the proteolytic processing sites," FEBS Lett. 207, 1-6 (1986)] This observation has been directly challenged by the introduction or deletion of residues believed to influence the adoption of such a conformation at a cleavage site within synthetic substrates. [Brakch et al., supra 1989; Gomez et al., supra 1989] Most recently a computer algorithm designed to predict the occurrence of "omega loops" (long unstructured loops) at known prohormone dibasic cleavage sites indicated that hydrolyzed bonds may indeed be associated with this conformational motic as well. [Bek, et al., "Prohormonal cleavage sites are associated with omega loops," Biochem. 29, 178-183 (1990)] However, the structural contributions made by the remaining regions of the polypeptide are virtually ignored, and may remain untested due to the difficulties encountered in manipulating large polypeptides.
One exemplary model of sequence-independent processing is the action of peptidases involved in the cleavage of signal peptides of secreted proteins and the leader sequences which specify the targeting of mitochondrial proteins. Comprehensive studies by von Heijne and other investigators have established the role played by particular residues positioned nearby the cleavage site in governing recognition by these endopeptidases. [von Heijne, et al., "Patterns of amino acids near signal-sequence cleavage sites," Eur. J. Biochem. 133, 17-21 (1983); Duffaud, et al., "Signal peptidases recognize a structural feature at the cleavage site of secretory proteins," J. Biol. Chem 263, 10224-10228 (1988); and Folz, et al., "Substrate specificity of eucaryotic signal peptidase," J. Biol. Chem. 263, 2070-2078 (1988)] However, amino acids far upstream the cleavage site both within the hydrophobic core domain and the positively charged amino terminal region characteristic of signal peptides of secreted proteins have been found to profoundly influence processing as well. Amino acid substitutions that distort the alpha-helical potential of pre-proparathyroid hormone signal peptide or place hydrophilic residues into the conserved hydrophobic domain yield poor substrates for the cleavage reaction catalyzed by the eukaryotic signal peptidase. [Caulfield, et al., "Synthetic substrate for eucaryotic signal peptidase," J. Biol. Chem. 264, 15813-15817 (1989)] Similarly, deletion of four residues from the amino terminus of yeast cytochrome oxidase subunit IV comprising the mitochondrial targeting signal served to prevent cleavage at the wildtype site twenty-five amino acids downstream. [Hurt, et al., "Amino-terminal deletions in the presequence of an imported mitochondrial protein block the targeting function and proteolytic cleavage of the presequence at the carboxy terminus," J. Biol. Chem. 262, 1420-1424 (1987)]
Magainins, first isolated as antibiotics, comprise a family of at least a dozen basic, ionophoric peptides and represents a model system for the study of vertebrate neuropeptides and hormones. [Zasloff, et al., "Antimicrobial activity of synthetic magainin peptides and several analogues," Proc. Natl. Acad. Sci. USA 85, 910-913 (1988); and Bevins, et al., "Peptides from frog skin," Annu. Rev. Biochem. 59, 395-414 (1990)] The magainin peptides are produced in the granular gland, specialized secretory cells that store large amounts of biologically active peptides and neurotransmitters. The granular glands are present in amphibian skin and stomach and release their contents in a holocrine fashion upon stress or injury. These structures are believed to serve physiological roles in defense against macroscopic predators and in microbial control following wounding. Strikingly, most hormones and many of the processing enzymes involved in their biosynthesis, stored in the anuran granular gland, have been found in the central nervous system and diffuse peripheral nervous system of mammals. [Bevins et al., supra (1990)]
The magainin peptides are synthesized from polyproteins, from which, in several cases, both antibiotic and hormonally active peptides are liberated. [Sures, et al., "Xenopsin: the neurotensin-like octapeptide from Xenopus skin at the carboxy terminus of its precursor," Proc. Natl. Acad. Sci. USA 81, 380-384 (1984); Richter, et al., "Sequence of preprocaerulein cDNAs cloned from skin of Xenopus laevis," J. Biol. Chem. 261, 3676-3680 (1986); and Poulter, et al., "Levitide, a neurohormone-like peptide from the skin of Xenopus laevis," J. Biol. Chem. 263, 3279-3283 (1988)] The primary sequences bracketing the biologically active peptides represent putative hormone processing sites, and these include the dibasic and monobasic cleavage sites characteristic of processing signals on mammalian neuropeptide precursors. The peptides contained within the granular gland are stored within secretory vesicles as processed, active species suggesting that initial proteolytic events occur prior to secretion. [Gibson, et al , "Novel peptide fragments originating from PGLa and the caerulein and xenopsin precursors from Xenopus laevis," J. Biol Chem. 261, 5341-5349 (1986)]
After peptides are discharged from the granular glands, they undergo further proteolysis, resulting in half-peptide fragments. Gibson et al., "Biosynthesis and degradation of peptides derived from Xenopus laevis prohormones," (1986); Giovannini, et al., supra (1987)] The half-peptide products have been fully characterized by extensive mass-spectroscopic analyses. Because the processed peptides no longer retain antibiotic activity, the processing reaction represents an inactivation step. At the same time, however, the half molecules accumulate in the secretion and several have been shown to undergo subsequent carboxyl-terminal amidation, a modification common to many hormones. [Gibson, et al., supra (1986)] Thus, although endoproteolysis inactivates the antibiotic activity of the magainin peptides, it may serve to liberate new hormones as well.
The secretions of Xenopus laevis has been used as a model to investigate, in vivo, the mechanisms of processing and biosynthesis of the peptide precursors and the fate of the peptides after secretion. [Giovannini et al., supra (1987)] Proteolysis of the larger primary products from X. laevis was reported to take place after secretion and possibly brought about by a cytoplasmic enzyme very specific for Xaa-Lys bonds, where Xaa is Ala, Lys, Leu or Gly. In the alternative it was postulated that proteolysis could be by an enzyme packed with the vesicles, but inactive before secretion. It was noted that xenopsin, containing the sequence Gly-Lys, was not cleaved under the reported conditions and it was suggested that the secondary structure of the peptides or neighboring amino acids play an important role in determining the accessibility of the site to proteolysis.
A wide variety of proteins can be and are currently produced by synthetic means such as recombinant technology. The generation of these recombinant proteins by host cells often result in proteins that require further processing to yield the mature functional protein desired. In attempts to mimic the natural processing of the recombinantly produced protein, the expressed proteins have been exposed to proteolytic enzymes. Generally, proteolytic enzymes are site specific and can result in multiple cleavages where the peptide of interest contains several sites recognized by the enzyme. In the field of synthetic protein production there is a need for enzymes with more sophisticated substrate specificity that can be utilized to process proteins such as the type that possess multiple recognition sites for traditional site specific enzymes.